Cytokines are intracellular signal transmitters which play an important role in an immune response, a response upon infection, hematopoiesis, inhibition of virus infection and tumor cells. Among them, a cytokine which transmits signals between lymphocytes is called interleikin (hereinafter, “IL”). Among ILs, IL-1 is a cytokine which mediates various immune responses and inflammatory responses, and is involved in maintenance of homeostasis of living organisms and produced from various cells such as monocytes, macrophages, keratinocytes, vascular endothelial cells and the like when the living organisms get infected or hurt. It has been known that there are two kinds of IL-1, IL-1α and IL-1β, both of which combine to the same receptor. It has been also known that IL-1 exerts its function simultaneously with the activation by an antigen to T cell and by mitogens, makes T cells release IL-2, and enhances the expression of IL-2 receptors to induce T cell proliferation, and that it acts on monocytes and macrophages in order to induce the production of TNF-α, IL-1, IL-6.
IL-1 has two kinds of IL-1 receptors (hereinafter “IL-1R”), and both of the IL-1Rs, type I and type II, have three immunogloblin-like domains in their extracellular domains. Type I receptors express in T cells and connective tissue, and type II receptors express in splenic B cells, myeloids and the like, and it has been known that type I receptors induce NF-κB in nuclei. It has been also known that there is an IL-1 receptor antagonist (hereinafter “IL-1ra”) which shows no bioactivity in spite that it binds to IL-1R with the affinity similar to that of IL-1α and IL-1β, and that it prevents IL-1 from binding to IL-1R competitively.
IL-18 is known to promote the production of interferon-γ (hereinafter “IFN-γ”), to enhance the activation of natural killer cells, to induce the production of IFN-γ from T cells in cooperation of IL-12, and to act an important role in a Th1 (IL-2 producting helper T cells) response. Further, it is known that IL-18 has no structural similarity to IL-12 in spite that it has a functional similarity, and has a structural similarity to IL-1. Moreover, it has been also known that IL-18 is produced as an inactive precursor that requires cleavage by IL-1β-converting enzyme (ICE)/caspasel for its maturation, as in the case of IL-1β, and that IL-18 activates IL-1R-associated kinase (IRAK) and NF-κB.
A plurality of molecules showing homology to IL-1R have been identified so far, and signal pathways mediated by IL-1R family is being studied intensively now. It has been known that MyD88 is a cytoplasmic protein comprised of an IL-1R homologous domain and a Death domain, and functions as an adaptor molecule which activates NF-κB by recruiting IRAK to IL-1R complex after IL-1 stimulation, and that MyD88 gene was originally separated as a myeloid differentiation primary response gene, which rapidly induces M1 myeloleukemic cells to macrophages by IL-6-stimulated differentiation.
Toxins in bacterial cells being comprised of lipopolysaccharide, which is a major structural component of the outer membrane encompassing peptidoglycan on the surface of Gram-negative bacteria, are called endotoxin, and it has been known that lipopolysaccharide is comprised of lipid called lipid A and various kinds of saccharide which covalently bind to the lipid A. It has been also known that this endotoxin has a bioactivity mainly involved in fever, decrease of leukocytes and platelet, hemorrhagic necrosis of bone marrow cells, hypoglycemia, induction of IFN, activation of B limphocyte (immune response cell derived from marrow), and the like.
It has been known that a Toll gene is required to control dorsoventral patterning during the embryonic development of Drosophila (Cell 52, 269–279, 1988, Annu. Rev. Cell Dev. Biol. 12,393–416, 1996), and for antifungal immune responses in adult fly (Cell 86, 973–983, 1996). It has been clarified that the Toll is a type I transmembrane receptor with an extracellular domain containing leucine-rich repeat (LRR) and that its cytoplasmic domain shows high homology to that of mammalian interleukin-1 recepter (IL-1R) (Nature 351,355–356, 1991, Annu. Rev. Cell Dev. Biol. 12, 393–416, 1996, J. Leukoc. Biol. 63, 650–657, 1998). It has been also clarified that another Toll family member, 18-wheeler, participates in the antibacterial host defense but not in the antifungal immune response, and that particular pathogens induce specific antimicrobial immune responses in Drosophila through the selective activation of the Tollpathways (Proc. Natl. Acad. Sci. USA 94, 14614–14619, 1997, EMBO J. 16, 6120–6130, 1997, Curr. Opin. Immunol. 11, 13–18, 1999).
Recently, mammalian homologs of Toll, designated as Toll-like receptors (TLRs), have been identified, and so far, six families including TLR2 and TLR4 have been reported (Nature 388, 394–397, 1997, Proc. Natl. Acad. Sci. USA 95, 588–593, 1998, Blood 91, 4020–4027, 1998, Gene 231, 59–65, 1999). It has been known that the TLR families, as in the case of the IL-1R, recruit IL-1R-associated kinase (IRAK) through the adaptor protein MyD88 as a signal transmitter and activate TRAF 6, and then activate NF-κB in the downstream (J. Exp. Med. 187, 2097–2101, 1998, Mol. Cell 2, 253–258, 1998, Immunity 11, 115–122, 1999). Further, the role of the TLR families in mammals is also believed to participate in innate immune recognition as pattern recognition receptors (PRRs), which recognize bacterial cell common structures (Cell 91, 295–298, 1997).
It has been reported that one of such pathogen-associated molecular patterns (PAMPs) to be recognized by the PRRs is lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria (Cell 91, 295–298, 1997), that said LPS stimulates host cells and makes them produce various proinflammatory cytokines including TNF-α, IL-1, and IL-6 (Adv. Immunol. 28, 293–450, 1979, Annu. Rev. Immunol. 13, 437–457, 1995), and that the LPS captured by LPS-binding protein (LBP) is delivered to CD14 on the cell surface (Science 249, 1431–1433, 1990, Annu. Rev. Immunol. 13, 437–457, 1995). However, since CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein without a transmembrane domain, the existence of a bona fide signaling receptor of LPS has been believed.
TLR4, which belongs to the TLR family, is a signaling molecule of LPS, which is a bacterial cell component of Gram-negative bacteria, and transfection of the TLR4 leads to a low constitutive activation of NFκB (J. Exp. Med. 188, 2091–2097, 1998, Nature 395, 284–288, 1998). On the other hand, as TLR2 transmits LPS signal when overexpressed in human embryonic kidney 293 cells in vitro, TLR2 has been thought to be a candidate for the LPS receptor. In addition, Godawski's group has reported that human TLR2 could interact with CD14 to form the LPS receptor complex (J. Immunol. 163, 639–643, 1999). Stimulation treatment with LPS leads to oligomerization of receptors and to subsequent recruitment of IRAK to the receptor complex. In contrast, groups of Poltorak and Qureshi have reported that TLR4 is the causative gene of the LPS hyporesponsiveness of C3H/HeJ mice, that is, the Lps gene, according to positional cloning (Science 282, 2085–2088, 1998, J. Exp. Med. 189, 615–625, 1999).
The inventors of the present invention have found by generation of TLR4-deficient mice that TLR4 is actually involved in LPS signaling (J. Immunol. 162, 3749–3752, 1999). The findings may be attributed to species-specific differences in the primary structure of TLR, in other words, LPS signaling could be mediated by TLR4 in mice and by TLR2 in humans. However, there is a report showing that mouse TLR2 also activated NF-κB in response to LPS (J. Immunol. 162, 6971–6975, 1999). In addition, Chow et al. have reported that they obtained the result showing that human TLR4 activated NF-κB-mediated gene expression by stimulation to LPS/CD14 in a dose-dependent or a time-dependent manner, which is consistent with the observation of C3H/HeJ mice, whereas they obtained the result conflicting with that of Kirschning's group when human 293 cells were used, and they have speculated that the differences of outcome may be due to differences in the lot of 293 cells as well (J. Biol. Chem. 274, 10689–10692, 1999).
Recently, it has been reported that TLR2 may not be involved exclusively in responsiveness to LPS derived from Gram-negative bacteria (J. Immunol. 162, 6971–6975, 1999) but may also act as a signaling receptor for peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria, which have another common bacterial structural pattern (J. Biol. Chem. 274, 17406–17409, 1999, J. Immunol. 163, 1–5, 1999). Further, it has been also reported that whole Gram-positive bacteria, soluble PGN, and LTA induced the activation of NF-κB in 293 cells expressing TLR2, but not induced the activation of NF-κB in the cells expressing TLR1 or TLR4 (J. Biol. Chem. 274, 17406–17409, 1999). Still further, it has been also reported that Chinese hamster ovary (CHO) fibroblast cells which express human TLR2 but not TLR4 were activated similarly by heat-killed Staphylococcus aureus and Streptococcus pneumoniae, and PGN derived from Staphylococcus aureus (J. Immunol. 163, 1–5, 1999).
Mycoplasmas, known as pathogens in animals and humans, are wall-less bacteria, yet they are capable of activating macrophages. A number of reports have identified this macrophage-activating material as lipoproteins/lipopeptides, and one of these lipopeptides, the 2 kD macrophage-activating lipopeptide MALP-2 derived from Mycoplasma fermentans, was biochemically fully characterized and has become available by synthesis (J. Exp. Med. 185:1951, 1997). It is known that the lipid moiety has 2 asymmetric C atoms, and that the synthetic MALP-2 comprised of the S, R racemate had a specific activation similar to the natural compound action at picomolar concentrations in vitro. Little is known about the signal pathways or the cell-surface receptors for MALP-2, except that MALP-2 activates NF-κB.
It is reported that lipoproteins/lipopeptides from mycobacterium and Borrelia burgdorferi induced the activation of host cells through TLR2 in vitro (Science 285, 736–739, 1999, Science 285, 732–736, 1999). Nevertheless, the conclusions obtained from overexpression experiments do not necessarily reflect the function of TLR family in vivo. It is also reported that the results of analysis of the responsiveness based on NF-κB activation are not related to biological responses mediated by these stimuli (Infect. Immun. 66, 1638–1647, 1998).
In addition, it is known that the function of a specific gene can be analyzed in individual level by using transgenic mice in which genes are artificially introduced and expressed, and gene-deficient mice generated by gene targeting in which specific genes on genomes are artificially transformed by homologous recombination with embryonic stem cells (hereinafter “ES cells”). In general, gene-deficient mice are called knockout mice, and TLR2 knockout mice and MyD88 knockout mice have not been known, and moreover, it has not been known that TLR2 knockout mice and MyD88 knockout mice are unresponsive to bacterial cell components, either.